My career objectives over the next five years are to obtain R01 funding, secure tenure at Delaware State University and develop a research niche in using Drosophila to elucidate the role of graded changes in acetycholine release on physiology and behavior during aging. As a graduate student with Janis O'Donnell (U. Alabama) and a post-doctoral fellow with David Krantz (UCLA), I received extensive training in Drosophila neurogenetics and on the study of neuronal processes relevant to human diseases. In addition, with Dr. Krantz, an expert in the study of vesicular transporters and their role in neurotransmission, I have developed my own expertise in the function of vesicular neurotransmitter transporters. My most recent projects have involved using Drosophila transporter to test gene-environment interactions that may contribute to the susceptibility to Parkinson's disease (PD). In particular, I determined that overexpression of the vesicular monoamine transporter (VMAT) can protect dopamine neurons from environmental toxins by sequestering cytosolic dopamine away from its site of action. To build on the potential neuroprotective role for VMATs, I conducted a screen for small molecules capable of increasing its function and I successfully identified a potentially new class of aminergic agents. I will continue to study the neurogenetics of Drosophila vesicular neurotransmitter transporters in the early stages of my career as an independent faculty, but change my primary focus from VMAT to the Drosophila vesicular acetylcholine transporter (VAChT). Under Dr. Krantz's excellent mentorship, I expanded my repertoire of skills to include cell culture, behavioral analyses, biochemical fractionation and pharmacology. I am now exploiting this background to determine the biological function for VAChT in my own lab as a PI. I have developed a rigorous training plan that will help me to achieve my career transition into the study of cholinergic neurotransmission during aging. One important aspect of my training during the K01 award will be to learn electrophysiology. I have received basic training from Diane O'Dowd, a specialist in adult Drosophila brain electrophysiology. I learned how to prepare fly brain tissue and identify Drosophila cholinergic neurons. In addition, I received input on general electrophysiological concepts and techniques from Felix Schweizer at UCLA. Under these two mentors, I was able to establish the basics of patching onto a neuron and recording action potentials. I now plan to master these foundational skills and become an expert. Both Drs. Schweizer and O'Dowd have offered to continue to support me with their expertise. Importantly, Dr. Melissa Harrington at Delaware State has offered to enhance my training and will serve as my hands-on primary physiology mentor. She has given me full access to her rig and under her mentorship I will continue to hone my skills. She has also provided substantial input into the current application. I recently started my own lab as an Assistant Professor in the NIH/COBRE Neuroscience program at Delaware State. I received a start-up package of $320,000. This support has afforded me valuable resources/personnel to achieve my goals (see Training Plan). My goal is to become an expert in using electrophysiology to investigate how differences in ACh release effect synaptic function. I will couple this skill set with my current expertise to determine how te changes in the function of cholinergic synapses may correlate with behavioral decline during aging. Moreover, I will be well-positioned to address fundamental questions about ACh release, behavior and aging that have clear translational significance. To study these issues, I will use an allelic series of point mutants in the Drosophila VAChT, the protein that packages and transports ACh for synaptic release. These alleles which range from mild to severe, and will allow me to determine the relationship between graded changes in acetylcholine release and progressive deficits in acetylcholine-mediated behaviors such as learning. The short life span of the fly coupled with additional genetic tools unique to this system will allow me to assess the impact of altered ACh release on behavior and synaptic physiology throughout the lifespan of the animal. In addition, I will test the hypothesis that an increase in cholinergic tone through overexpression of VAChT will improve age-related deficits in cognitive functions. In future aims, I will use the model system I propose here as a platform to identify small molecules capable of increasing ACh release. I believe that these agents could represent a new therapeutic strategy to improve the cognitive decline associated with decreased ACh neurotransmission in both normal and pathological aging.